SPRAY FREEZING

Abstract

The present invention relates to an improved method for preservation of e.g. microorganisms, especially lactic acid bacteria, said method includes spray freezing.

Claims

1. A process for preserving microorganisms (esp. LAB (Lactic Acid Bacteria)), such as preserving by freezing and drying, comprising the following steps: a) preparing droplets of an aqueous (or liquid) suspension containing the microorganisms, e.g. by spraying the suspension; and b) contacting the droplets with a drying gas; and c) contacting the droplets resulting from the preceding step with a cryogenic gas; and d) optionally subjecting the resulting frozen product from the preceding step to a further drying step, such as drying under reduced pressure, e.g. freeze-drying; and e) optionally packaging the microorganisms, such as in an air-tight and/or moisture-tight package.

2. A process for drying a microorganism (esp. a LAB) containing suspension, characterized in that: c) an aqueous suspension containing microorganisms is sprayed into a cryogenic gas in a spray chamber; and d) the frozen product from the preceding step is collected and freeze dried until a water activity of less than 0.20 is achieved.

3. A process for drying a microorganism (esp. a LAB) containing suspension, characterized in that: b) an aqueous suspension containing microorganisms is sprayed into a drying gas in a spray chamber; c) the product from the preceding step is contacted with a cryogenic gas in a chamber; and d) the frozen powder from the preceding step is collected and freeze dried until a water activity of less than 0.20 is achieved.

4. A process for drying (or removing liquid from) a solution or suspension containing proteins (such as enzymes, hormones, human proteins, therapeutically active proteins or lipoproteins) or microorganisms (such as bacteria, LAB, yeasts, fungi, plant cells, animal cells, or vira), characterized by: a) Preparing droplets of the suspension or solution, e.g. by spraying the solution or suspension; and b) contacting the droplets with a drying gas; and c) contacting the droplets obtained in step b) with a cryogenic gas; and d) subjecting the resulting frozen product from the preceding step to a further drying step, such as drying under reduced pressure, e.g. freeze-drying; and e) optionally packaging the product, such as in an air-tight and/or moisture-tight package.

5. A process for freezing a suspension containing microorganisms (esp. LAB), by: Spraying the suspension into a chamber containing a drying gas; and Freezing the product from the preceding step by contacting with a cryogenic gas in a chamber; and Optionally packaging the frozen suspension, such as in an air-tight and/or moisture-tight package.

6. The process of any preceding claim, wherein the spraying is carried out by means of a spray nozzle (atomizing device), such as an ultrasound nozzle; a pressure nozzle; a two-fluid nozzle (e.g. using N2 as atomizing gas); or a rotating atomizing device, the atomizing preferably resulting in droplets having a size from 10 to 500 micrometer, such as having a size selected from one of the following ranges: 15 to 400, 20 to 350, 50 to 350, 100 to 350, 20 to 300, 20 to 200, 50 to 300, 50 to 200, 100 to 300, or 100 to 200, measured as Dv90 values in micrometer.

7. The process of any preceding claim, wherein the frozen product (e.g. powder from the freezing step) is collected by means of a cyclone (preferably with a maximum differential pressure drop across the cyclone of approx. 100 mm water column), or an electrostatic filter.

8. The process of any preceding claim, characterized in that the spray drying step takes place at a pressure in the range of 60 to 200 kPa, such as in the range 80 to 150 kPa, or 60 to 120 kPa, 70 to 110 kPa, or 105 to 140 kPa.

9. The process of any preceding claim, characterized in that the freezing step takes place at a pressure in the range of 60 to 200 kPa, such as in the range 80 to 150 kPa, or 60 to 120 kPa, 70 to 110 kPa, or 105 to 140 kPa.

10. The process of any preceding claim, characterized in that the final drying step (of the frozen product) takes place under reduced pressure, such as by freeze-drying, preferably to an aw below 0.20.

11. The process of any preceding claim, wherein the “retention time” is less than 2 minutes in the spray dryer, and preferably the resulting spray-dried (or partly dried) product is directly introduced into a freezing chamber, such as by using an apparatus wherein the dried (or partially dried) product from the spray drying is transferred into the freezing chamber by means of the gravity.

12. The process of any preceding claim, wherein the spray-drying step is carried out at with a drying gas inlet temperature of at most 300° C., advantageously between 20° C. and 250° C. and more preferably between 100° C. and 200° C.

13. The process of any preceding claim, wherein the spray-drying step takes place at a temperature in the range from 20° C. to 250° C., such as in the range 30 to 230° C. or in the range 60 to 200° C.

14. The process of any preceding claim, wherein after the spray-drying step, the droplet has a size of between 20 and 400 microns and preferably between 50 and 300 microns.

15. The process of any preceding claim, wherein after the spray-drying step the liquid (e.g. water) content of the droplet is at least 5% by weight reduced compared to the starting suspension/solution, preferably at least 10%, at least 0%, at least 40% or at least 60%.

16. The process of the preceding claim, wherein after the spray-drying step the liquid (e.g. water) content of the droplet is between 20% and 85% by weight, (preferably between 30% and 80%, or between 40% and 75% percent by weight), with respect to the total weight of the droplet.

17. The process of any preceding claim, wherein the duration of the freeze-drying step is a time suitable for obtaining a powder whose residual water content of the powder is at least 0.15.

18. The process of any preceding claim, wherein the drying gas contains less than 5% oxygen, such as less than 2%.

19. The process of any preceding claim, wherein the cryogenic gas contains less than 5% oxygen, such as less than 2%.

20. The process of any preceding claim, wherein the drying gas is selected from the group consisting of an inert gas (such as Nitrogen), a noble gas (such as Helium, Argon or Neon) etc., carbon dioxide, and an alkane gas (such methane), and a mixture thereof.

21. The process of any preceding claim, wherein the cryogenic gas is selected from the group consisting of an inert gas (such as nitrogen), a noble gas (such as helium, argon or neon) etc., carbon dioxide, and an alkane gas (such methane), and a mixture thereof.

22. The process of any preceding claim, wherein the cryogenic gas has an inlet temperature in the range from −50 to −250° C. or in the range from −80 to −160° C., and/or the cryogenic gas has a temperature or between −20° C. and 150° C., or between −50° C. and −100° C. during the freezing step.

23. The process of any preceding claim, wherein the solution or suspension further comprises an additive that stabilizes the material/microorganism.

24. The process of the preceding claim, wherein the additive is selected from the group consisting of: Inositol, lactose, sucrose, trehalose, inulin, maltodextrin, skimmed milk powder, yeast extract, casein peptone, inosine, inosinemonophospate, glutamine and salts thereof (such as monosodium glutaminate), casein or salts thereof (such as sodium caseinate), ascorbic acid and salts thereof (such as sodium ascorbate), and polysorbate.

25. The process of the preceding claim, wherein the ratio heat labile material (protein or microorganism):additive is within the range from 1:0.1 to 1:10 or from 1:0.5 to 1:5, such as from 1:1 to 1:4 or from 1:1½ to 1:3, (w/w of the dry weights).

26. The process of any preceding claim, wherein the microorganism is selected from the group consisting of: a yeast (e.g. Saccharomyces), a lactic acid bacterium, a Streptococcus species, a Lactobacillus species, a Lactococcus species, a Leuconostoc species, a Bifidobacterium species, an Oenococcus species, and a Bacillus species.

27. The process of any preceding claim, wherein the microorganism is selected from the group consisting of Streptococci species, such as Streptococcus thermophilus.

28. The process of any preceding claim, wherein the microorganism is selected from the group consisting of Bifidobacterium species, such as B. animalis ssp. lactis or B. longum.

29. The process of any preceding claim, wherein the microorganism is selected from the group consisting of Lactobacillus species, such L. acidophilus or L. bulgaricus.

30. The process of any preceding claim, wherein the microorganism is selected from the group consisting of Lactococcus species, such as L. lactis or L. cremoris

31. The process of any preceding claim, wherein the microorganism is selected from the group consisting of Bacillus species, such as B. subtilis.

32. The process of any preceding claim, wherein the protein is a metabolite of a Bacillus species, such as a metabolite of B. subtilis or B. amyloliquefaciens.

33. The process of any preceding claim, wherein the protein is an enzyme (such as an aspartic protease (e.g. a chymosin), a protease or a lactase or a lipase).

34. The process of any preceding claim, wherein the protein is a milk protein, such as whey or casein.

35. The process of any preceding claim, wherein the protein is a therapeutically active protein, such as a hormone, insulin, a growth factor, or an antibody.

36. A product obtainable by the process of any preceding claim.

37. The product of the preceding claim, which is packaged (e.g. in an airtight container).

38. An apparatus usable in the process of any preceding claim, such as an apparatus comprising a chamber with i) an atomizing means for atomizing the suspension, ii) an inlet for a drying gas (optionally integrated in the atomizing means), iii) an inlet for a cryogenic gas, and iv) an outlet optionally connected with a cyclone, e.g. an apparatus substantially as depicted on FIG. 1 or FIG. 4.

39. The apparatus of the preceding claim, which comprises a two-chamber tower (wherein the first chamber is placed over the second chamber) wherein the first (upper) chamber (11) comprises i) an atomizing means for atomizing the suspension (5), and ii) an inlet for the drying gas (optionally integrated in the atomizing means); and the second (lower) chamber (12) comprises i) an inlet for a cryogenic gas and ii) an outlet coupled to a first cyclone (14).

40. An apparatus (such as a spray tower) comprising an first (upper) chamber (11) and a second (lower) chamber (12), wherein the upper chamber comprises means for atomizing a suspension or solution (5); and an inlet for a drying gas (1), and means (3) for heating the drying gas to a temperature in the range 20° C. to 250° C.; and wherein the lower chamber comprises an inlet for a cryogenic gas (4) (adapted for a gas having a temperature in the range −50 to −250° C.) and optionally a tank for storing the cryogenic gas; and an outlet for the frozen product, said outlet being connected to a cyclone (13); and wherein the upper chamber is placed so the (partly) dried particles drop into the lower chamber for subsequent freezing.

41. An apparatus, such as an apparatus according to the preceding claim, comprising an first (upper) chamber and a second (lower) chamber, wherein the upper chamber comprises means for atomizing a suspension or solution; and means of injection of drying gas; and means for supplying the drying gas at a temperature in the range 20° C. to 250° C.; And wherein the lower chamber comprises means for injection of a cryogenic gas; and means for supplying the cryogenic gas at a temperature in the range −50 to −250° C.; and an outlet for the frozen product, said outlet preferably being connected to a cyclone.

42. The apparatus of any preceding claim, wherein the first chamber is connected to a heater for heating the drying gas.

43. The apparatus of any preceding claim, wherein the second chamber is connected to a tank adapted to a cryogenic gas.

44. The apparatus of any preceding claim, which comprises means for lowering the pressure (e.g. to a pressure below 0.9 bar) in the first chamber and/or in the second chamber.

45. The apparatus of any preceding claim, which comprises means for increasing the pressure (e.g. to a pressure above 1.1 bar) in the first chamber and/or in the second chamber.

46. The apparatus of any preceding claim, wherein the upper chamber has a height that allows at least 5% of the liquid in the suspension/solution to evaporate during the passage, and wherein the lower chamber has a height that allows a complete freezing of the product entering from the upper chamber.

47. The apparatus of any preceding claim, wherein the first chamber is essential cylindrical and has a diameter of 0.5 to 5 m and a height of 1 to 4 times the diameter.

48. The apparatus of any preceding claim, wherein the second chamber is essential cylindrical and has a diameter of 0.5 to 5 m and a height of 1 to 2 times the diameter.

49. The apparatus of any preceding claim, wherein the first and second chamber is connected so the first chamber is the upper part of and the second chamber is the lower part of an essential cylindrical structure which and has a diameter of 0.5 to 5 m and a total height of 2 to 6 times the diameter.

50. Use of the apparatus of any preceding claims, wherein a drying gas (having a temperature in the range 20° C. to 250° C.) and a liquid containing a protein or a microorganism is sprayed into the upper chamber; and a cryogenic gas (having a temperature in the range −50 to −250° C.) is sprayed into the lower chamber.

51. The process of any of claims 1 to 35, which process takes place in an apparatus according to any of claims 38 to 49.

52. The process of any of claims 1 to 35, which process takes place in an apparatus substantially as depicted in the FIGS. 1, 4, 5, and/or 6.

Description

FIGURES

[0100] FIG. 1 depicts a preferred spray drying equipment that can be used according to the invention.

TABLE-US-00001 (a) drying gas supply (b) drying gas heater (c) Inlet temperature control loop (d) Combined spray drying/freezing chamber (e) Liquid feed supply (f) Liquid feed pump (g) Atomization device (h) Cyclone separator (i) Warm water scrubber unit (j) Exhaust fan (k) Chamber pressure control loop (l) Outlet temperature control loop (m) powder discharge (y) cryogenic gas inlet

[0101] FIG. 2 depicts the stability data for the strain ST-44 after 3 months storage at −20° C. and at +5° C., cf. example 4.

[0102] FIG. 3 depicts the stability data for the strain ST-4895 after 3 months storage at −20° C. and at +5° C., cf. example 2.

[0103] FIG. 4 depicts a spray drying/freezing apparatus according to the invention

TABLE-US-00002 1. Drying gas supply 2. Supply fan 3. Heater 4. Cryogenic gas supply 5. Nozzle (with optionally gas supply shown) 6. Liquid feed 7. Liquid feed tank 8. Protein or Microorganism suspension, optional with cryoprotectant 9. Water inlet 10. Liquid feed pump 11. Drying chamber 12. Freezing chamber 13. Frozen powder discharge 14. Cyclone 15. Exhaust fan 16. Exhaust gas T. Temperature regulator P. Pressure regulator F. Drying gas regulator

[0104] FIGS. 5 and 6 depicts preferred embodiments of the apparatus

EXPERIMENTAL

Example 1

[0105] A sample of 1281 g of Streptococcus thermophilus (strain ST-Fe 2) concentrate was kept at <5° C. This contained 1.7E+11 CFU/g with approx. 12.8% (w/w) dry solids. Parallel to this 579 g of solution was prepared by adding the following ingredients to 470 g of cold tap water (approx. 10° C.) under agitation: 33 g sodium ascorbate, 32 g sodium caseinate, 22 g inositol and 22 g monosodium glutamate (MSG).

[0106] The sample and the additive solution were mixed. This resulted in 1.86 kg of liquid formulation with approx. 14.6% (w/w) dry solids to be spray dried. This liquid formulation contained now approx. 1.2E+11 CFU/g and was kept cold (<5° C.) throughout the test.

[0107] A GEA Niro Mobile Minor laboratory spray dryer was modified to accommodate spray drying using two 380 mm top extension sections followed by liquid nitrogen injection in the lower fixed section of the standard spray chamber to accommodate in-situ freezing of the partially dehydrated product droplets arriving from the upper section of the chamber. The upper spray drying section was supplied with heated pure nitrogen drying gas and the lower freezing section was supplied with liquid nitrogen capable of generating a frozen particulate colder than −100° C.

[0108] The upper spray dryer section inlet temperature was kept at 190° C., using a nitrogen drying gas kept at a mass flow-rate of approx. 100 kg/h. A 2-fluid nozzle (Schlick 0-2) was used for the atomization of the above mentioned liquid formulation, using an atomization gas flow of approx. 5 kg/h (Nitrogen) equivalent to an atomization pressure of 0.8 Bar(g)

[0109] The liquid formulation was sprayed into the upper spray dryer section. The feed-rate was kept at 2 kg/h and the spray drying/freezing chamber outlet temperature was kept in the range −140 to −110° C.

[0110] A free-flowing frozen powder with an average particle size of 105 micron was collected below the downstream cyclone. After 55 min. about 1100 g of partially dehydrated frozen formulation has been collected, which corresponds to a yield of about 70%. The moisture content was now 18.5% (w/w) measured as total volatiles on a Sartorious IR at 115° C. This corresponds to a reduction of the total water amount in our product of approx. 24% (w/w).

[0111] The obtained partially dehydrated frozen powder contained now 1.5E+11 CFU/g. The frozen powder was freeze dried, performed at a chamber pressure of 0.3 mbar with temperature increasing from −42° C. to 32° C. with 1.5° C./min. The freeze drying was ended when the weight of the product has been constant/stable for at least 2 hours. The dried product had an acceptable stability after 3 months storage at 5° C. (pH 5.6 as measured using standard CINAC analysis).

Example 2

[0112] Example 1 was repeated using the same equipment, conditions and additive solution, but with the strain ST-4895. Thus a sample of 1281 g Streptococcus thermophilus strain ST-4895 concentrate was mixed with 579 g of additive solution, resulting in 1.86 kg of liquid formulation with approx. 14.6% (w/w) dry solids to be spray dried. This liquid formulation contained now approx. 1.2E+11 CFU/g. After drying and freezing, a frozen powder was obtained.

[0113] The spray-frozen powder was freeze dried (cf. example 1). The stability of the dried product was compared with a freeze-dried product obtained from a “standard” pellet-frozen concentrate of ST-4895. Performance of the freeze dried products was examined by using standard CINAC analysis. For three months stability data, see FIG. 3. The product produced by the process according to present invention is stable, even if compared with the pellet-frozen product.

Example 3

[0114] Example 1 was repeated using the same equipment, conditions and additive solution, but with the Streptococcus thermophilus strain ST-143. Thus, a sample of 1281 g of Streptococcus thermophilus (strain ST-143) concentrate was mixed with 579 g of additive solution. This resulted in 1.86 kg of liquid formulation with approx. 14.6% (w/w) dry solids to be spray dried. This liquid formulation contained now approx. 1.2E+11 CFU/g. After drying and freezing, a frozen powder was obtained.

Example 4

[0115] Example 1 was repeated using the same equipment, conditions and additive solution, but with the Streptococcus thermophilus strain ST-44. Thus, a sample of 1281 g of strain ST-44 concentrate was mixed with 579 g of additive solution.

[0116] This resulted in 1.86 kg of liquid formulation with approx. 14.6% (w/w) dry solids to be spray dried. This liquid formulation contained now approx. 1.2E+11 CFU/g. After drying and freezing, a frozen powder was obtained.

[0117] The spray-frozen powder was freeze dried as in example 1, and the stability of the dried product was compared with a product obtained by freeze drying a pellet-frozen concentrate of ST-44 (method as in example 2). For three months stability data, see FIG. 2. The product produced according to the present invention is stable, even if compared to the pellet-frozen product.

Example 5

[0118] A sample of 2640 g of Bifidobacterium animalis ssp. lactis (strain BB-12®) concentrate was kept at <5° C. This contained 2E+11 CFU/g with approx. 14.5% (w/w) dry solids. Parallel to this 1080 g of solution was prepared by adding the following ingredients to 876 g of cold tap water (approx. 10° C.) under agitation: 60 g sodium ascorbate, 79 g skimmed milk powder, 33 g inositol and 33 g MSG. The sample and the additive solution were mixed. This resulted in 3.72 kg of liquid formulation with approx. 15.7% (w/w) dry solids to be spray dried. This liquid formulation contained now approx. 1.4E+11 CFU/g and was kept cold (<5° C.) throughout the test. After drying and freezing preformed as in example 1, a frozen powder was obtained. The frozen powder was freeze dried, and the dried product had an acceptable cell count after 3 months storage at 5 C (2.9E+11 CFU/g).

Example 6

[0119] A sample of 1145 g of Lactobacillus bulgaricus (strain LB CH-2) concentrate was kept at <5° C. This contained 1.1E+11 CFU/g with approx. 11.5% (w/w) dry solids. Parallel to this 375 g of solution was prepared by adding the following ingredients to 282 g of cold tap water (approx. 10° C.) under agitation: 27 g sodium ascorbate, 36 g skimmed milk powder, 15 g inositol and 15 g MSG. The sample and the additive solution were mixed. This resulted in 1.52 kg of liquid formulation with approx. 14.7% (w/w) dry solids to be spray dried. This liquid formulation contained now approx. 8.5E+10 CFU/g and was kept cold (<5° C.) throughout the test. After drying and freezing preformed as in example 1, a frozen powder was obtained. The frozen powder was freeze dried, and the dried product had an acceptable stability after 3 months storage at 5 C (pH 6 as measured using standard CINAC analysis).

REFERENCES

[0120] EP1234019B1 (Danisco A/S) [0121] U.S. Pat. No. 6,010,725A (Nestle SA) [0122] Semyonov et al (Food Research International 43, 193-202 (2010) [0123] U.S. Pat. No. 7,007,406 (Wang) [0124] WO15063090A2, WO14029758A1, WO14029783A1 (Chr Hansen A/S) [0125] ISO 13320:2009 standard for Particle size analysis—Laser diffraction methods

[0126] All references cited in this patent document are hereby incorporated herein in their entirety by reference.